Transferrin is an iron binding glycoprotein that transports iron in blood plasma and in the extracellular fluid of tissues. Transferrin binds two ferric (Fe+3) ions per molecule with high affinity, and normally all iron in plasma is bound to transferrin. The trivalent iron is delivered to the cell cytoplasm via transferrin receptors located on the cell surface. Apotransferrin is the iron-free transferrin; iron-saturated transferrin is referred to as Holotranferrin. As used in the present context, the term “transferrin” designates any form of transferrin, irrespective of its iron content.
Medical use of transferrin relates to the following main phenomena:
Pathological conditions in which the concentration of iron exceeds the iron binding capacity of transferrin, and non-transferrin-bound-iron can be detected in serum samples of the patients. Such pathological conditions include malignant neoplastic diseases, e.g. leukemia. The appearance of non-transferrin-bound-iron is particularly common during cytotoxic chemotherapy of malignant diseases (Reviewed in Bear and Steward, 1996 Lancet 347: 342-343). Transferrin may therefore be used for the prevention of the harmful effect of non-transferrin-bound-iron in patients (U.S. Pat. No. 6,251,860).
Transferrin receptors, located on the cell surface membrane, may be utilized for targeting compound into the cells, using transferrin-compound conjugates. It has been recently found that transferrin receptors are located on tumor cells, and therefore transferrin conjugates are designed for the diagnosis, imaging, localization and treatments of tumors (e.g. U.S. Pat. Nos. 5,000,935; 4,886,780; 4,895,741). Transferrin conjugates are also used for targeting nucleic acids into the cell (U.S. Pat. No. 5,792,645) and for the treatment of intracellular pathogens that reside in phagosomes (U.S. Pat. No. 6,054,133).
A growing need for serum-free cell culture media is raised by the pharmaceutical industry. Transferrin products are widely used as cell growth factors in such cultures.
Transferrin is also necessary as an additive to some cell cultures, including cultures of embryonic stem cells even in the presence of serum.
In any aspect of the pharmaceutical use, the transferrin to be used must meet high standards of purity and sterility, while maintaining maximum biological activity, designated by the iron-binding capacity.
Methods of obtaining purified, virally inactivated, non-toxic transferrin are known in the art U.S. Pat. No. 4,841,026 describes a method for viral inactivation using pasteurization for 10 hours at 60° C. As plasma proteins, including transferrin, are heat-sensitive they must be heat-inactivated in conjugation with stabilizers. The method disclosed therein therefore comprises the saturation of Cohn fraction containing transferrin with an excess of iron followed by the removal of free iron radicals and unwanted protein via filtration and ion exchange chromatography. The iron-saturated transferrin is then pasteurized. The method is therefore suitable only for the production of holo-transferrin; moreover, purity and bioactivity are not disclosed. In U.S. Pat. No. 5,252,715 a purified transferrin is pasteurized in the presence of complexing agent, and the complexing agent is removed with the bound iron, resulting in iron-free transferrin In this patent, aggregates produced during the heat treatment are removed by aluminum hydroxide, which is not desired in a preparation for pharmaceutical use.
U.S. Pat. No. 5,041,537 discloses a method of preparing high purity transferrin comprising the steps of precipitating the γ-globulins from the fraction containing the transferrin, removing the precipitate from residual liquid by ultrafiltration or gel filtration and concentrating the filtrate to the desired protein and ion concentration. Virus inactivation is obtained by a procedure that comprises UV radiation in the presence of β-propiolactone or treating the solution with specific detergent followed by ion-exchange chromatography. However, the transferrin obtained shows iron binding capacity of only about 80%. U.S. Pat. No. 5,744,586 describes a method for obtaining at least 95% pure transferrin. As described therein, a partially purified plasma fraction containing transferrin is concentrated and its ionic strength is reduced. The transferrin-containing fraction is chemically treated to inactivate enveloped viruses, and applied thereafter to an ion exchange medium in which the transferrin is adsorbed. The fraction comprising transferrin is then eluted from the ion exchange column and further nanofiltered to remove non-enveloped viruses.
U.S. Pat. No. 6,251,860 describes a pharmaceutical composition containing pure apotransferrin, having iron binding capacity of at least 90%, free from polymers and containing a maximum 3% of dimers. The method for virus inactivation includes two different ion exchange chromatography steps with solvent/detergent treatment, followed by filtration through a virus removal filter. Purity disclosed in this patent is at least 98%; however, purity is measured as the percentage of β-globulin and not as transferrin.
In summary, although prior art disclosures provide various methods for the production of transferrin, each method focuses on one or a few aspects of the product (purity, virus contamination, bioactivity, apo- or holo-transferrin etc.).
Therefore, there is a recognized need for, and it would be highly advantageous to have ultra-pure, virus safe, commercially reproducible, apo- and holo-transferrin.